AAGPs TM Anti-Aging Glyco Peptides. Enhancing Cell, Tissue and Organ Integrity Molecular and biological attributes of lead AAGP molecule

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1 AAGPs TM Anti-Aging Glyco Peptides Enhancing Cell, Tissue and Organ Integrity Molecular and biological attributes of lead AAGP molecule 1

2 Acknowledgements This presentation was prepared by Dr. Samer Hussein and based on the results of testing conducted on the family of AAGPs created by Dr. Geraldine Deliencourt- Godefroy. Dr. Hussein received his Ph.D. from McGill University in Montreal. His expertise is in the area of Molecular and Cellular biology with an emphasis on the signals required to direct early stem cells to differentiate into neural cells. His main focus is on reprogramming skin fibroblasts into induced pluripotent stem cells (ips cells). He works at Samuel Lunenfeld Research Institute at Mount Sinai Hospital in Toronto as a Post-doctoral Fellow in the Nagy Lab. Dr. Deliencourt-Godefroy received her Ph.D. from the University of Rouen, France. She is the Founder of TF Chem, a drug discovery company which uses the fluorine atom properties to develop new glycomimetic compounds: the GlycoMim technology. TF Chem is a leader in the area of fluorinated glycosides and their applications as new, more stable and potent and safer therapeutics. Dr. Deliencourt-Godefroy has received a number of awards: Vermeil Medal of Innovation and Research, Francinov (Versailles); Award from the Contest of Innovative and Technological Companies of the French Ministry of Research and Technology; Award from the Tremplin Enterprise competition from the French Senate. 2

3 Agenda! ProtoKinetix and the development of AAGP TM! Molecular Structure! Testing and Evaluation of AAGP TM effects on cellular viability! Functional Studies using AAGP TM 3

4 A look at ProtoKinetix Inc.! Biotechnology Company Research Focus! Development and testing of a family of patented Anti-Aging Glycopeptides (AAGPs) which enhance cell and tissue survival against environmental stresses! Commercial Targets! Cell and tissue protection! Stem cell preservation! Cell therapy and regenerative medicine! Organ Transplants! Blood and blood derivative preservation! Anti-aging for skin care/cosmetics 4

5 Structure of Lead AAGP Molecule! Several molecules have been studied and tested! Example of Lead Structure! Ala-Ala-Lys-F-C-F-Galactose! Formula C20H35CF2N4O11! Molecular Mass Daltons! Highly water soluble 650 grams/liter! Peptides in the structure suggests lipid solubility 5

6 History behind AAGP TM! AAGPs were derived from work on Anti- Freeze Glycoproteins (AFGPs)! AFGPs are natural proteins produced in living organisms such as fish, plants, and insects! AFGPs enable survival under freezing temperatures! They protect the cellular membrane from freezing damage 6

7 History behind AAGP TM (cont.)! Research on AFGPs showed great promise.! Some research areas! Preservation of biological materials! Food preservative! Organ transplantation! Large scale applications of AFGPs are impractical! Costly extraction from unsustainable sources! Very large molecule in its native forms (2,600 33,000 Daltons)! Cannot be purified beyond 85%! Solution: Development of AFGP synthetic analogs 7

8 History behind AAGP TM (cont.)! AFGP analogs! Unstable! Very susceptible to degradation! Limited bioactivity! Large complex molecules (2,600 33,000 Daltons)! AAGP Technology! Stable! Not susceptible to degradation! Highly bioactive! Small Molecule ( Daltons) Summary: New proprietary stable analogs found to have cell survival and anti-aging properties 8

9 Agenda! ProtoKinetix and the development of AAGP TM! Molecular structure! Testing and Evaluation of AAGP TM effects on cellular viability! Functional Studies using AAGP TM 9

10 Stability Evaluation of AAGP TM! AAGP TM remains highly stable under the following conditions:! ph range: ~2 (highly acidic) to ~14 (highly alkaline)! No enzymatic degradation from:! Proteases (peptide cleaving enzymes)! Galactosidases (sugar degrading enzymes)! Temperature! Range: normal body temperature (37 C) to cryotemperatures(-197 C) 10

11 Toxicity Evaluation of AAGP TM! Non-toxic to cells in vitro (up to 5 mg/ ml tested)! Transplanted AAGP treated islet cells in vivo (mice) showed no toxicity 11

12 Bioactivity Testing of AAGP TM! AAGP TM demonstrated an unprecedented spectrum of applicability under harsh cellular stresses! Cellular Stress Tests:! Nutrient Deprivation! Temperature and Cryopreservation! Oxidative Stress from Hydrogen Peroxide (H 2 O 2 )! UV Radiation! Inflammation 12

13 Nutrient Deprivation AAGP maintains viability of adult skin fibroblasts during nutrient depletion and nutrient deprivation Cell Viability (%) 160% 140% 120% 100% 80% 60% 40% 20% 0% Control AAGP d0 d1 d2 d3 d4 d0 d1 d2 d3 d4 Low Serum Conditions (minimal nutrients) Starvation Conditions (no nutrients) 13

14 Bioactivity Testing of AAGP TM! Cellular Stress Tests:! Nutrient Deprivation! Temperature and Cryopreservation! UV radiation! Oxidation stress by Hydrogen Peroxide (H 2 O 2 )! Inflammation 14

15 Protection from Moderate to Cold Temperatures AAGP maintains 100% viability of adult skin fibroblasts under moderate and cold temperatures 3 C Survival Rate (%) 140% 120% 100% 80% 60% 40% 20% 0% Control AAGP 15 C 140% * *** *** *** *** ** 120% 100% 80% 60% 40% 20% 0% d1 d2 d3 d4 d5 d1 d2 d3 d4 d5 Survival Rate (%) Control AAGP *** *** *** *, p< 0.05; **, p< 0.01; ***, p< Viability Test: Trypan Blue Exclusion 15

16 AAGP maintains stem cell survival at 3 C AAGP enhances survival of mescells at low temperatures Cells were placed at 3 C for either 24 or 48 hrs., then at 37 C for an additional 24hrs prior to cell counting, as a measure of colony formation capacity post 3 C. Media: ES qualified media Viability assay: Trypan Blue Exclusion 24 hrs Survival Rate (%) 48 hrs Survival Rate (%) 400% 300% 200% 100% 0% 40% 30% 20% 10% 0% Mouse ES cells at 3 C Control AAGP 10,000 50, ,000 Initial Cell Density (Cells/mL) Control AAGP 10,000 50, ,000 Initial Cell Density (Cells/mL) 16

17 Human ES Cell Cryopreservation AAGP enhances cryo-preservation of Human Embryonic Stem Cells regardless of their density 62% 58% AAGP s effect is concentration 60% dependent 40% Cell Viability (%) 120% 100% 80% 40% Human ES Cells Cryopreservation Control 76% 1 mg/ml AAGP 2.0 mg/ml AAGP 5 mg/ml AAGP 49% 87% 71% 74% 20% 0% 2.2 Million cells/ml 3.2 Million cells/ml 17

18 Effect of AAGP on Cord Blood Cryopreservation AAGP (1mg/ml) AAGP treatment doubles the CD34 + stem cell count within Cord Blood samples post cryopreservation AAGP s effect is concentration dependent Optimal Concentration: 4.0 mg/ ml % Change (AAGP relative to Dextran Control) 250% 200% 150% 100% 50% 0% Total Nucleated Cells Progenitor (CD34+) cells AAGP (2mg/ml) AAGP (4mg/ml) Colony Forming Units 18

19 AAGP during Cryo-preservation *, p < 0.05; **, p < 0.01; ***, p < AAGP enhances cryopreservation of mammalian cell lines with DMSO Effect is consistent across all cell lines tested AAGP concentration: 2mg/ml 10% DMSO freezing media Cell Viability (%) 120% 100% 80% 60% 40% 20% *** 53% Control * 68% 79% AAGP 93% 78% * 93% 78% 0% 24% mes Skin Fibroblasts HEK293 Hela 19

20 Bioactivity Testing of AAGP TM! Cellular Stress Tests! Nutrient Deprivation! Temperature and Cryopreservation! UV radiation! Oxidative Stress from Hydrogen Peroxide (H 2 O 2 )! Inflammation 20

21 AAGP protects against UVA Skin Fibroblasts 105% 100% **, p< % Control Cell Viability (%) 85% 75% 88% 87% 83% 83% 75% ** 70% AAGP-1 10 J/cm2 10 J/cm2 + AAGP-1 65% 59% 55% "-24h" 0h 1h 24h 21

22 Protection against UVC Viable Cells (%) AAGP added immediately BEFORE irradiation AAGP added immediately AFTER irradiation 180% 160% 140% 120% 100% 80% 60% 40% 20% Control AAGP Control AAGP 140% ** p < 0.01 p< % * 150% 120% 143% 104% 100% 100% 117% 117% 100% 80% 64% 60% 64% 51% 40% 40% 29% UVC: 400J/m 2 29% UVC: 400J/m 2 20% Viable Cells (%) 0% 0h 24h 48h 96h AAGP protects adult skin fibroblasts against UVC when added before and after irradiation 0% 0h 24h 48h 96h AAGP treatment post irradiation suggests a repair effect 22

23 Protection against Oxidative Stress 1mM H 2 O 2 at 37 C 10hrs Effects of AAGP on H 2 O 2 treated Neonatal Skin Fibroblasts at 37 C Control AAGP AAGP Control Cell Viability (%) 120% 100% 80% 60% 40% 20% 0% RED: Dead cells/eth-br derivative GREEN: Live cells/calcein 0h 2h 10h 23

24 Bioactivity Testing of AAGP TM! Cellular Stress Tests! Nutrient Deprivation! Temperature and Cryopreservation! UV radiation! Oxidation stress by Hydrogen Peroxide (H 2 O 2 )! Inflammation 24

25 Anti-Inflammatory Effect AAGP protects against in vitro IL 1-! induced inflammatory response (Cox2 expression) Cell line: Hela cells Quantification from RT-PCR analysis * p < 0.05 * 25

26 Agenda! ProtoKinetix and the development of AAGP TM! Testing and Evaluation of AAGP TM effects on cellular viability! Functional Studies using AAGP TM 26

27 Functional assays on Platelets and Islets Cells Platelets Preservation and Aggregation Islet cells Diabetes and Islet cell transplantation 27

28 Preservation of Platelets Untreated 22 C 1 mg/ml AAGP 4 mg/ml AAGP Untreated platelets, no AAGP added, show strong and consistent aggregation In the presence of AAGP, platelet activation is prevented (i.e. low aggregation count) 15 C 4 C 28

29 AAGP Treated Islet Cell Transplantation AAGP increases viable mouse islet cells within the transplant Model: Streptozocin-induced diabetic mouse Method: Islet cell transplantation from AAGP treated and untreated islets Measure of response: Blood Glucose Level Islets treated with AAGP stabilized blood glucose level in diabetic mice much more efficiently than controls Results by Dr. Ray V. Rajotte, University of Alberta * * * * untreated AAGP * * 29

30 AAGP Presentation Summary Lead AAGP molecule is stable and bioactive It has protective properties for cells and platelets under multiple stress conditions It enhances their viability and preserves their function, both in vitro and in vivo These protective properties may be extended to tissues and organs in general AAGP technology has potential applications in a variety of therapeutic areas involving cell, tissue and organ preservation and possibly preserving body tissue integrity 30

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